Vaccination is widely accepted as the favoured approach to tackle the global healthcare burden of infectious disease and cancer. However, despite significant advances in our understanding of the molecular biology relating to infectious disease and cancers, the development of effective vaccines in these areas has been limited. The most effective vaccines developed use live, attenuated organisms, however, the safety risk associated with such attenuated pathogens reverting to virulence has restricted their widespread use. A further major barrier preventing the wide scale development and use of more effective vaccines is the limited ability to identify candidate pathogen derived proteins that will elicit broad protective immunity in a specific manner against variant strains of microbial pathogens.
One particular approach that shows the promise of conferring broad, protective immunity is the use of stress protein complexes as vaccines against infectious disease and cancer (Colaco et al., (2004) Biochem Soc Trans 32:626-628 and Zeng et al., (2006) Cancer Immunol Immunother 55:329-338). It has also been widely documented that heat shock protein/antigenic peptide complexes are efficacious as vaccines against specific cancers (U.S. Pat. No. 5,997,873; U.S. Pat. No. 5,935,576, U.S. Pat. No. 5,750,119, U.S. Pat. No. 5,961,979 and U.S. Pat. No. 5,837,251). It has been shown that pathogen derived stress protein complexes isolated from heat-shocked BCG cells induced T-helper 1 (Th1) lymphocyte mediated immune responses in a vaccinated host, which conferred protective immunity against a live challenge in a murine aerosol challenge model of pulmonary tuberculosis (International PCT Patent Application No. WO 01/13944). Moreover, it has been shown in WO 02/20045, WO 00/10597 and WO 01/13943 that stress protein complexes isolated from pathogens or pathogen infected cells are effective as the immunogenic determinant within vaccines against infectious diseases.
Heat shock proteins (hsps, HSPs) form a family of highly conserved proteins that are widely distributed throughout the plant and animal kingdoms. On the basis of their molecular weight, the major heat shock proteins are grouped into six different families: small (hsp20-30 kDa); hsp40; hsp60; hsp70; hsp90; and hsp100. Although heat shock proteins were originally identified in cells subjected to heat stress, they have been found to be associated with many other forms of stress, such as infection, osmotic stress, cytokine stress and the like. Accordingly, heat shock proteins are also commonly referred to as stress proteins (SPs) on the basis that their expression is not solely caused by a heat stress. Members of the hsp60 family include the major chaperone GroEL. These form multimeric complexes with co-chaperones such as GroES. Many microbial pathogens have additional hsp60 families that form distinct complexes from GroEL and some hsp60 family members may be more immunogenic, such as the hsp65 of mycobacteria. Members of the hsp70 family include DnaK which can form multimeric complexes with co-chaperones such as DnaJ. Other major hsps include the AAA ATPases, the Clp proteins, Trigger factor, Hip, HtpG, NAC, Clp, GrpE, SecB and prefoldin.
Stress proteins are ubiquitously expressed in both prokaryotic and eukaryotic cells, where they function as chaperones in the folding and unfolding of polypeptides. A further role of stress proteins is to chaperone peptides from one cellular compartment to another and, in the case of diseased cells, stress proteins are also known to chaperone viral or tumour-associated peptides to the cell-surface.
The chaperone function of stress proteins is accomplished through the formation of complexes between stress proteins and the chaperoned polypeptide. Chaperoned polypeptides may include peptide fragments, with the formation of such complexes controlled by an ATP-dependent nucleotide exchange system, which has been most clearly demonstrated for the bacterial Hsp70 homologue, DnaK (Szabo et al. PNAS (1994) Vol 91. 10345-10349). Briefly, in its resting cellular state, DnaK is bound to ATP (adenosine triphosphate) and has a low affinity for substrate (Palleros et al. PNAS (1991) Vol. 88. 5719-5723). ATP hydrolysis results in conversion of DnaK to a high-affinity ADP (adenosine diphosphate) state, resulting in the formation of DnaK-ADP-substrate complexes, where the substrate is typically a polypeptide or protein. Following ADP dissociation, ATP re-binds to DnaK, resulting in a conformational change that triggers the release of the correctly folded substrate protein from the complex (Palleros et al J Biol Chem (1992) Vol 267, No 8, 5279-5285; Palleros et al. Nature (1993) 365(6447):664-6; Szabo et al. PNAS (1994) Vol 91. 10345-10349). This final step, resulting in release of substrate, has been shown to require potassium (K+) and magnesium (Mg2+) in addition to the binding of ATP (Palleros et al. Nature (1993) 365(6447):664-6; Palleros et al. FEBS Letters (1993) Vol 336, No 1, 124-128).
Heterologous polypeptides or polypeptide fragments complexed with the stress proteins form stress protein-peptide complexes, which may be referred to as heat shock protein complexes (HspCs). HspCs are captured by antigen presenting cells (APCs) to provide a source of antigenic peptides which can be loaded onto major histocompatibility complex (MHC) molecules for cell surface presentation to the T lymphocytes of the immune system.
Heat shock protein/antigenic peptide fragment complexes (HspCs) have been widely studied as cancer vaccines (see, for example U.S. Pat. No. 5,997,873 and U.S. Pat. No. 5,935,576) and methods have thus been developed for the isolation of HspCs from tumour cells for use as effective vaccines against such tumours. For example, WO 02/28407 discloses a method for use in purifying protein complexes based on the binding affinity of heat shock proteins for heparin. The two step approach involves heparin affinity chromatography and a subsequent ion exchange chromatography step which is optional in order to obtain the stress protein complex preparations. WO 02/34205 relates to the purification of HSP70 stress protein complexes using Con A Sepharose. These methods however result in the isolation of individual families of heat shock proteins, and therefore neglect the use of multiple chaperone proteins as vaccines.
The use of HspCs as cancer vaccines can be significantly improved by the use of multiple chaperone proteins, in particular heat shock proteins (Bleifuss et al 2008) and thus methods have been developed for the purification of multiple chaperone proteins and chaperone protein complexes for use in vaccines. For example U.S. Pat. No. 6,875,849 discloses the use of free-solution isoelectric focusing (FS-IEF) for the purification of HspCs from tumours for use as cancer vaccines. Free flow isoelectric focusing (FF-IEF) can be used to isolate heat shock protein/peptide complexes from pathogens and infected cells for use as the immunogenic determinant in vaccine compositions for the prevention and treatment of infectious diseases. However, a key limitation of that technique has been the difficulties associated with developing a large scale FF-IEF instrument to produce the quantities of heat shock protein/peptide complexes (HspCs) which would be required for large, commercial scale, GMP vaccine manufacture. Moreover, the use of ampholytes (ampholines) to produce the pH gradient required during the FF-IEF process results in the introduction of a further contaminant, in addition to the chaotropes, in the resulting, purified HspC containing preparations. Such contaminants, being unacceptable to Regulatory Authorities, pose a significant barrier to the use of FF-IEF methodology in the manufacture of HspC containing vaccine compositions. Interestingly, these inventors report the stability of HspCs even in the presence use of the chaotropes, such as urea and detergents used during FF-IEF purification even in the absence of divalent cations and ADP in the process buffers (Bleifuss et al 2008). Additionally, the process of free-flow isoelectric focussing is slow with a typical run time of 4 hours, during which high levels of protein degradation result, severely limiting the use of FF-IEF in large scale production of purified protein complexes.